UC-NRLF 


Influence  of  Some  Organic  Compounds 
upon  the  Hydrolysis  of  Starch 
by  Salivary  and  Pancre- 
atic Amylases 


r       t 
t  ,L  •  • 


BY 


NELLIE  M.  NAYLOR 


DISSERTATION 

SUBMITTED  IN  PARTIAL  FULFILLMENT  OF  THE  RE 
QUIREMENTS  FOR  THE  DEGREE  OF  DOCTOR  OF 
PHILOSOPHY  IN  THE  FACULTY  OF  PURE 
SCIENCE,  COLUMBIA  UNIVERSITY 


Reprinted  from  the  Journal  of  the  American  Chemical  Society,  Vol.  44,  pp.  2957-66. 


NEW  YORK 
1922 


•-. 


\ 


Influence  of  Some  Organic  Compounds 
upon  the  Hydrolysis  of  Starch 
by  Salivary  and  Pancre- 
atic Amylases 


BY 

NELLIE  M.  NAYLOR 


DISSERTATION 

SUBMITTED  IN  PARTIAL  FULFILLMENT  OF  THE  RE- 
QUIREMENTS FOR  THE  DEGREE  OF  DOCTOR  OF 
PHILOSOPHY  IN  THE  FACULTY  OF  PURE 
SCIENCE,  COLUMBIA  UNIVERSITY 


Reprinted  from  the  Journal  of  the  American  Chemical  Society,  Vol.  44,  pp.  2957-66. 


NEW  YORK 
1922 


ACKNOWLEDGMENTS 

This  investigation  was  undertaken  at  the  suggestion  of  Professor  H.  C. 
Sherman,  and  was  carried  out  under  his  direction.  The  author  wishes  to 
express  to  Professor  Sherman  her  appreciation  of  his  advice  and  encour- 
agement received  throughout  this  work. 

The  author  also  wishes  to  thank  Professor  A.  W.  Thomas  and  Dr. 
Mary  L.  Caldwell  for  helpful  suggestions  and  advice. 


EXCHANG* 


INFLUENCE    OF    SOME    ORGANIC    COMPOUNDS    UPON   THE 

HYDROLYSIS   OF   STARCH   BY   SALIVARY  AND 

PANCREATIC  AMYLASES 

Various  organic  compounds  have  been  reported  as  influencing  the  ac- 
tivity of  amylases  in  the  digestion  of  starch.  In  18931  and  again  in  1904s, 
Effront  investigated  the  effects  of  certain  amines  and  amino  acids  upon 
the  hydrolysis  of  starches  by  an  infusion  of  malt  extract.  Glycine,  alanine, 
leucine,  glutamic  acid,  hippuric  acid,  creatine,  creatinine,  asparagine  and 
aspartic  acid  were  found  to  increase  the  activity  of  the  amylase,  while 
amides  and  aliphatic  amines  appeared  to  act  as  inhibitory  agents.  Ford 
and  Guthrie,3  using  Lintner  soluble  starch  with  malt  extract  and  with  a 
purified  malt  amylase,  studied  the  effect  of  asparagine,  glycine  and  alanine 
on  the  starch  digestion.  They  ascribe  the  apparent  increase  of  activity 
in  the  presence  of  the  amino  acids  to  the  amphoteric  property  of  these 
compounds  or  to  their  effect  in  neutralizing  some  inhibitory  impurity  in 
the  starch  or  enzyme  solution. 

Terroine  and  Weill4  tested  the  influence  of  many  of  the  amino  acids  on 
the  saccharification  of  starch  by  pancreatic  juice.  They  report  an  ac- 
tivating influence  on  the  part  of  the  amino  acids  tested,  but  they  make 
no  statement  regarding  the  hydrogen-ion  concentration,  or  regarding  the 
purity  of  any  of  the  materials  used. 

Desgrez  and  Moog5  report  an  activating  influence  exerted  by  methyl, 
ethyl  and  trimethyl  amine  hydrochlorides,  on  the  hydrolysis  of  starch  by 
a  glycerol  extract  of  dry  pancreas.  As  in  the  work  reported  by  previous 
investigators,  there  is  no  indication  of  any  tests  for  hydrogen-ion  concen- 
tration or  for  purity  of  materials  used.  Nor  is  there  any  evidence  that 

1  Effront,  Mon.  Sci.,  41,  266  (1893). 

2  Bffront,  ibid.,  61,  561  (1904). 

8  Ford  and  Guthrie,  /.  Chem.  Soc.,  89,  76  (1906). 

4  Terroine  and  Weill,  Compt.  rend.  soc.  bioL,  72,  542  (1912). 

6  Desgrez  and  Moog,  Compt.  rend.,  172,  553  (1921). 


6 

the  influence  of  simple  inorganic  salts  was  provided  for,  so  that  so  far  as 
their  data  show,  the  activation  may  have  been  due  simply  to  the  effects 
of  the  substances  as  chlorides  rather  than  to  the  organic  radicals. 

Rockwood6  studied  a  large  number  of  nitrogen  compounds  with  regard 
to  their  influence  on  the  hydrolysis  of  starch  by  saliva.  Those  compounds 
which  seemed  to  increase  the  activity  of  the  amylase  were  called  auxo- 
amylases.  It  was  reported  that:  (1)  a-amino  acids  act  as  auxo-amylases, 
as  evidenced  by  the  effect  of  added  glycine,  tyrosine  and  aspartic  acid; 
(2)  anthranilic  acid,  and  its  meta  and  para  isomers,  in  which  the  amino 
group  is  not  alpha  to  the  carboxyl  group,  were  also  reported  to  be  auxo- 
amylases;  but  sulfanilic  acid,  in  which  the  carboxyl  group  is  replaced  by 
the  group,  SC^OH,  was  not;  (3)  the  substitution  of  one  hydrogen  of  the 
amino  group  by  benzoyl,  as  in  hippuric  acid,  did  not  destroy  the  activating 
properties ;  (4)  amines  of  the  methane  series  were  reported  to  act  as  auxo- 
amylases;  (5)  amides  were  not  auxo-amylases.  The  results  of  Rockwood's 
experiments  are,  however,  not  conclusive,  since  several  factors  now  known 
to  influence  the  activity  of  the  amylase  were  not  standardized  and  appar- 
ently not  taken  into  account.  As  mentioned  in  connection  with  other 
work,  there  is  no  evidence  that  the  influence  of  inorganic  salts  was  pro- 
vided for;  also  the  hydrogen-ion  concentration,  determined  as  "neutral 
to  litmus,"  would  be  only  approximately  known,  and  not  necessarily  that 
at  which  optimum  activity  of  the  amylase  is  obtained.  Therefore  the 
"activation"  reported  may  be  due  to  the  influence  of  other  factors,  in- 
stead of  to  the  effect  of  the  organic  groupings  in  the  compounds  tested. 

In  this  Laboratory  the  work  of  testing  the  influence  of  amino  acids  on 
the  hydrolysis  of  starch  by  amylases  has  been  done  under  much  more 
closely  standardized  conditions;  the  starch  and  all  salts  used  were  purified, 
the  hydrogen-ion  concentrations  of  all  starch  dispersions  tested,  either 
electrometrically  or  colorimetrically  with  standardized  buffer  mixtures, 
and  the  enzyme,  either  purified  or  natural,  was  always  present  in  a  starch 
paste  "activated"  by  an  optimum  concentration  of  sodium  chloride  and 
sodium  phosphate.  Under  these  conditions,  it  has  been  determined7 
that  neutralized  aspartic  acid  and  asparagine,  glycine,  alanine,  phenyl- 
alanine  and  tyrosine  increase  the  saccharogenic  activity  of  saliva,  pan- 
creatin,  and  purified  pancreatic  and  malt  amylases.  It  was  also  deter- 
mined8 that  glycine,  phenylalanine,  arginine  and  cystine  increase  the 
amyloclastic  activity  of  purified  pancreatic  amylase,  while  histidine  and 
tryptophane  do  not  show  this  influence. 

It  was  the  purpose  of  this  investigation  to  study  the  influence  of  certain 
organic  compounds,  containing  typical  groupings,  on  the  hydrolysis  of 

6  Rockwood,  J.  Am.  Chem.  Soc.,  39,  2745  (1917). 

7  Sherman  and  Walker,  (a)  ibid.,  41,  1866  (1919);  (b)  43,  2461  (1921). 

8  Sherman  and  Caldwell,  ibid.,  43,  2469  (1921). 


starch  by  amylases,  to  see  whether  this  might  throw  some  light  upon 
the  problem  of  whether  the  favorable  effect  of  amino  acids  is  due  to  a 
direct  activation  attributable  to  their  organic  structure,  as  considered  by 
Rockwood,  or  is  due  to  the  conservation  of  the  enzyme,  as  brought  out 
by  previous  work  in  this  Laboratory, 7b-8  or  due  to  both.  It  was  planned 
to  use  several  simple  organic  compounds,  in  which  the  influence  of  the 
carboxyl  group  alone,  the  amino  group  alone,  and  the  carboxyl  and  amino 
groups  in  the  same  molecule,  could  be  studied,  and  then  to  extend  the 
investigation  to  the  influence  of  other  groupings,  especially  those  present 
in  the  amino  acids  which  have  been  tested  in  this  Laboratory.  Several 
of  the  compounds  used  in  this  work  have  been  studied  by  other  investi- 
gators, but  since  the  results  were  inconclusive,  as  has  been  pointed  out, 
the  work  has  been  repeated  by  the  standardized  method76  used  in  this 
Laboratory  for  testing  the  influence  of  the  amino  acids  on  the  hydrolysis 
of  starch  by  jamylases.  The  compounds  were  chosen:  (1)  to  contain  the 
amino  group  in  aniline  sulf ate  and  in  methyl  and  ethyl  amine  hydrochlor- 
ides,  the  carboxyl  group  in  benzoic  acid,  and  the  amide  group  in  benz- 
amide;  (2)  to  show  the  effect  of  the  position  of  the  amino  group  by  study- 
ing glycine,  in  which  the  amino  group  is  alpha  to  the  carboxyl  group,  and 
anthranilic  acid  in  which  the  amino  group  is  ortho  to  the  carboxyl  group; 
(3)  to  show  .the  effect  of  substitution  of  the  hydrogen  of  the  amino  group, 
as  in  hippuric  acid;  (4)  to  test  the  influence  of  indole  and  guanidine,  and  to 
compare  their  effect  with  that  of  the  amino  acids  containing  these  groupings. 

Materials  Used 

Lintner  soluble  starch  was  purified  by  repeated  washings  with  distilled  water  and 
with  thrice  distilled  water.  The  starch  was  air-dried  and  the  moisture  was  determined. 
The  acidity  was  determined  by  electrometric  titration  of  a  1  %  starch  dispersion  con- 
taining the  amounts  of  sodium  chloride  and  disodium  phosphate  used  for  pancreatic 
amylase  work.8  All  water  used  in  making  starch  pastes  and  activating  solutions,  and  in 
the  final  rinsing  of  glass  ware,  was  distilled  from  alkaline  permanganate,  then  from  dil. 
phosphoric  acid,  through  a  block-tin  condenser,  and  collected  in  Non-sol  bottles,  in 
which  it  was  kept  until  used. 

The  sodium  chloride  and  sodium  phosphate  used  as  activating  agents  were  re- 
crystallized  twice  from  distilled,  and  once  from  thrice  distilled  water,  air-dried,  and  an- 
alyzed for  moisture.  All  of  the  organic  substances  employed  were  carefully  purified 
and  tested  for  purity. 

Experimental  Procedure 

The  equivalent  of  10  g.  of  dry  starch  was  weighed,  mixed  with  cold  water,  poured 
into  boiling  water,  and  the  mixture  boiled  for  3  minutes.  This  was  cooled,  made  up 
to  a  volume  of  250  cc.,  and  allowed  to  settle.  Twenty-five-cc.  portions  of  this  starch 
dispersion  were  introduced  into  lOOcc.  cylinders  and  the  required  amount  of  0.01  N 
sodium  hydroxide  solution  was  added.  The  activating  agents,  sodium  chloride  and 
sodium  phosphate,  in  amounts  previously  determined  for  the  enzyme  to  be  used9  were 
added  to  the  cylinders. 


9  Sherman  and  Kendall,  /.  Am.  Chem  Soc.,  32,  1087  (1910). 


8 

The  substances  to  be  tested  in  this  work  were  in  most  cases  difficultly  soluble,  and 
more  or  less  subject  to  hydrolysis.  The  amount  to  be  used  for  several  cylinders  was 
weighed  out,  water  and  the  small  quantity  of  sodium  hydroxide  (0.01  N  or  0.02  N) 
or  hydrochloric  acid  (0.01  N)  required  for  neutralization  were  added  to  the  substance,  and 
carefully  warmed  not  above  40°,  until  the  substance  was  dissolved.  This  solution  was 
cooled,  and  made  up  to  a  definite  volume,  and  portions  to  represent  the  desired  quantity 
of  the  substance  were  added  to  the  cylinders,  from  a  buret.  The  contents  of  each 
cylinder  was  then  made  up  to  a  total  volume  of  100  cc.,  stirred,  and  the  cylinders  were 
placed  in  a  thermostat  regulated  at  40d=0.01°.  While  the  cylinders  were  attain- 
ing the  temperature  of  the  surrounding  water,  the  enzyme  solution  was  prepared, 
and  introduced  into  clean,  dry  flasks.  The  activated  starch  dispersion  at  40°  was  added 
to  the  enzyme,  the  whole  thoroughly  mixed  and  allowed  to  react  for  exactly  30  minutes, 
the  flasks  being  kept  at  constant  temperature.  The  effect  of  light  was  excluded  by  work- 
ing in  a  north  room  with  window  shades  drawn,  and  avoiding  the  use  of  any  artificial  light 
during  digestion.  The  reaction  was  stopped  by  the  introduction  of  Fehling  solution, 
and  the  flasks  were  placed  in  a  boiling  water  bath  for  15  minutes.  The  amount  of 
cuprous  oxide  formed  by  the  reducing  sugar  present  was  determined. 

Since  sodium  sulfate  would  be  present  in  every  test  in  which  neutralized  aniline 
sulfate  was  used,  any  difference  in  the  activity  of  the  enzyme  might  be  due  to  the  effect 
of  the  aniline  or  to  the  sodium  sulfate.  When  sodium  sulfate  was  tested  in  amounts 
corresponding  to  those  which  would  be  formed  in  the  solutions  containing  neutralized 
aniline  sulfate,  it  was  found  to  have  no  influence  on  the  activity  of  the  amylases  used. 

Since  earlier  work10  has  shown  that  the  optimum  activity  of  pancreatic  amylase  is 
obtained  for  only  a  small  range  of  hydrogen-ion  concentration,  it  is  necessary  to  deter- 

TABLE  I 
LOG  Cu+  IN  MOLES  PER  LITER  FOUND  IN  DIGESTION  MIXTURES  USED 

0.01  M  solution  added  except  with  benzoic  acid  and  benzamide,  of  which  50  mg.  each  was 

added 
Starch  Paste  (activated)  Log  CB+  Log  CH+ 

(electrometric)  (colon  metric) 

No  substance  added —6 .94  —6 .93 

Benzoic  acid —6 .96  —6 .93 

Benzamide —6.90  —6.93 

Aniline" -6.87  -6.93 

Hippuric  acid ....  —6 .93 

Anthranilic  acid ....  —6 .93 

Methyl  amine  hydrochloride -6 .90  -6 .93 

Ethyl  amine  hydrochloride —6 .89  —6 .93 

Indole -6 .87  -6 .93 

Guanidine  hydrochloride —6 .88  —6 .93 

Tryptophane -6 .96  -6 .93 

Alanine -6.86  .-6.93 

0  Aniline  sulfate,  calculated  to  the  desired  amount  of  aniline,  was  used  in  all  cases. 

mine  accurately  the  values  for  each  digestion  mixture  used.  These  determinations 
were  made  by  electrometric  titration  (if  possible)  of  the  substance  to  be  tested,  in  a 
buffered  starch  dispersion;  then  in  each  "set"  the  hydrogen-ion  concentration  was 
checked  by  a  colorimetric  test  of  the  starch  dispersion  actually  used  in  digestion,  or  that 
of  a  separate  cylinder  made  up  exactly  like  the  one  used.  In  all  colorimetric  tests  the 

10  Sherman,  Thomas  and  Baldwin,  J.  Am.  Chem.  Soc.,  41,  231  (1919). 


9  •••••::/•: 

color  comparison  was  made  with  a  standardized  buffer  solution,11  using  bromothymol 
blue  as  indicator,  range  PH  6.0  to  7.6.  The  results  of  these  hydrogen-ion  determinations 
are  given  in  Table  I. 

In  order  to  prove  that  hydrolysis  of  the  substances  tested  did  not  change  the  hydro- 
gen-ion concentration  of  the  mixtures  during  digestion,  colorimetric  or  electrometric 
tests  were  made  of  the  starch  dispersions,  before  and  after  digestion.  In  all  cases,  where 
solutions  were  buffered  with  sodium  phosphate,  the  hydrogen-ion  concentration  was 
found  to  be  constant  throughout  the  experiment. 

Data  of  Typical  Experiments 

Table  II  shows  the  influence  of  equimolar  quantities  of  methyl  and  ethyl 
amine  hydrochlorides,  aniline,  anthranilic  acid,  hippuric  acid  and  glycine, 
when  tested  in  the  presence  of  activating  salts,  sodium  chloride  and  sodium 
phosphate,  and  when  tested  in  the  absence  of  these  salts. 

TABLE  II 

OF  EQUIMOI,AR  AMOUNTS  OF  CERTAIN  ORGANIC  SUBSTANCES  ON  THE  HY- 
DROLYSIS OF  STARCH  BY  SALIVA 

In  presence 

of  salts  In  absence  of  salts 

Cc.  of  pure  saliva  per  100  cc.  of 

starch  dispersion 
Added  material  0.4  0.4  0.8 


0.01  M 
None                                  .    .  . 

Cuprous 
oxide 

Mg. 

294 

Cuprous 
oxide 

Mg. 

2  6 

Cuprous 
oxide 
Mg. 

Methyl  amine  hydrochloride 

297 

281  0 

Kthyl  amine  hydrochloride  

293 

285.0 

None 

208 

0  5 

4  6 

Aniline  

202 

3.0 

25.0 

Anthranilic  acid  

191 

6.0 

15.0 

None 

197 

2  0 

4  5 

Hippuric  acid  , 

180 

1.0 

22.0 

Glvcine.  . 

209 

0.3 

4.3 

This  experimental  work  shows  that  very  small  amounts  of  reducing  sugar 
are  obtained  by  the  action  of  saliva  on  a  pure  starch  dispersion,  in  the 
absence  of  added  electrolytes,  as  has  also  been  found  to  be  true  in  the 
case  of  the  pancreatic  amylase,  even  when  tested  in  the  form  of  commercial 
pancreatin.12  The  presence  of  0.01  M  ethyl  and  methyl  amine  hydro- 
chlorides,  in  the  media  in  which  no  other  salts  are  added,  activates  the 
enzyme  to  such  an  extent  that  almost  as  much  reducing  sugar  is  obtained 
as  in  the  presence  of  sodium  chloride  and  sodium  phosphate.  Since  aniline, 
tested  as  sulfate,  does  not  show  this  effect,  the  influence  exerted  by  methyl 
and  ethyl  amine  hydrochlorides  cannot  properly  be  interpreted  as  showing 
any  specific  effect  of  the  amino  group,  but  is  probably  due  to  the  favorable 

11  W.  M.  Clark,  "Determination  of  Hydrogen  Ions,"  Williams  and  Wilkins  Co., 
1920,  p.  81. 

12  Sherman  and  Schlesinger,  /.  Am.  Chem.  Soc.,  34,  1104  (1912);  37,  1305  (1915). 


10 

influence  upon  the  amylase  of  the  chloride  ions  thus  introduced  into  the 
digestion  mixture.9 

Anthranilic  acid,  hippuric  acid  and  glycine  tested  in  the  absence  of 
inorganic  salts,  showed  no  influence  on  the  rate  of  digestion  of  starch  when 
0.4  cc.  of  pure  saliva  per  100  cc.  of  starch  dispersion  was  used.  However, 
when  the  concentration  of  enzyme  was  doubled  in  the  starch  dispersions 
containing  these  substances,  no  inorganic  salts  being  added,  thus  making 
the  conditions  comparable  with  the  experiments  described  by  Rockwood, 
an  activating  influence  was  obtained.  This  activation  may  be  attributed 
to  the  presence  of  a  larger  amount  of  electrolyte  in  the  increased  volume 
of  saliva  used,  rather  than  to  the  effect  of  the  substances  added.  This 
view  is  supported  by  the  fact  that  when  anthranilic  acid,  hippuric  acid 
and  aniline  are  tested  in  the  presence  of  sodium  chloride  and  sodium 
phosphate,  they  show  no  "activating"  influence.  These  experiments 
confirm  the  statements  already  made,  that  tests  for  the  influence  of  any 
substances  on  the  digestion  of  starch  by  saliva  or  by  pancreatic  amylase 
must  be  made  in  the  presence  of  inorganic  salts;9  and  indicate  that  the 
"activation"  reported  by  Rockwood  and  other  investigators  as  attributable 
to  organic  structure  is  misleading,  and  is  probably  due  to  the  influence  of 
the  added  substances  upon  hydrogen-ion  or  electrolyte  concentration 
rather  than  to  any  specific  effect  of  the  organic  groups. 

In  order  to  determine  whether  the  organic  groups  discussed  by  Rock- 
wood  have  activating  effects  upon  salivary  and  pancreatic  amylases  when 

TABLE  III 

INFLUENCE  OP  BENZOIC  ACID,  ANILINE  SULFATE,  BENZAMIDE,  ANTHRANILIC  ACID  AND 
HIPPURIC  ACID  ON  THE  HYDROLYSIS  OF  STARCH  BY  SALIVA  AND  BY  PURIFIED  PANCREATIC 

AMYLASE 
Material  added  Saliva      Pancreatic  amylase 

Cuprous  oxide         Cuprous  oxide 
Mg.  Mg.  Mg. 

None 327  298 

Benzoic  acid 50  324  274 

Benzoic  acid 100  321  272 

None '.         ...  331  282 

Hippuric  acid 50  322  261 

Hippuric  acid 100  314  254 

None 218  217 

Aniline 50  217  206 

Aniline 100  225  203 

None 296  312 

Benzamide 50  289  310 

Benzamide 100  289  308 

None 331  317 

Anthranilic  acid 50  313  305 

Anthranilic  acid 100  319  305 

these  are  tested  in  the  presence  of  the  usual  "activating"  salts,  a  series  of 
experiments  was  carried  out  with  saliva  and  pancreatic  amylase  tested 


in  the  presence  and  absence  of  benzoic  acid,  hippuric  acid,  aniline  sulfate, 
benzamide  or  anthranilic  acid. 

The  technique  of  these  experiments  was  the  same  as  has  already  been 
described,  the  mixtures  always  being  "activated"  by  chloride  and  phos- 
phate and  made  up  to  the  correct  hydrogen-ion  concentration  for  the 
enzyme  to  be  used.  Typical  results  are  shown  in  Table  III.  The  re- 
sults obtained  with  benzoic  acid  and  with  aniline  sulfate  (Table  III) 
and  methyl  and  ethyl  amine  hydrochlorides  (Table  II)  on  the  hydrol- 
ysis of  starch  by  amylases,  are  taken  as  typical  of  the  influence  of  the 
carboxyl  and  amino  groups,  when  tested  under  conditions  suitable  to  the 
normal  activity  of  the  enzyme.  Since  no  favorable  effect  is  shown  by  any 
of  these  compounds,  the  presence  of  the  carboxyl  group  alone  or  the  amino 
group  alone  does  not  account  for  the  "activation"  of  the  amylases  by  amino 
acids.  Benzamide  shows  no  effect  on  the  activity  of  the  amylases  used. 
Glycine,  as  well  as  most  other  natural  ammo  acids,  has  been  shown  to 
give  definite  "activation"  of  the  amylases  in  the  digestion  of  starch, 7b  while 
anthranilic  acid,  containing  both  the  amino  and  carboxyl  groups  does  not 
activate. 

Since  in  these  experiments  only  the  a-amino  acids,  such  as  glycine,  and 
other  products  of  protein  hydrolysis  have  been  found  to  increase  the 
activity  of  the  amylases,  it  is  plain  that  neither  the  amino  nor  the  car- 
boxyl group  alone,  nor  the  presence  of  both  in  the  same  molecule  is  suffi- 
cient to  induce  any  "activating"  influence  upon  the  amylases  when  present 
in  a  substrate  solution  containing  proper  amounts  of  simple  electrolytes. 

One  may,  therefore,  conceive  either  that  the  "activating' '  effects  of  amino 
acids,  like  glycine,  are  due  to  their  structural  configuration  in  that  they 
contain  amino  radicals  in  the  a  position  to  the  carboxyl  group,  or  that 
their  favorable  influence  is  due  to  the  conservation  of  the  enzyme  through 
diminution  of  its  hydrolytic  destruction  in  the  water  solutions  in  which 
it  acts.7b  It  has  not  been  feasible  to  test  a-amino  acids  known  not 
to  be  products  of  protein  hydrolysis  because  our  knowledge  of  the  hydro- 
lytic products  of  the  proteins  is  not  yet  sufficiently  complete.  It  might, 
perhaps,  be  expected  that  if  true  "activation"  can  be  attributed  to  a-amino 
acid  structure,  per  se,  it  should  not  be  entirely  lost  in  a  derivative  such 
as  hippuric  acid  (benzoyl  glycine).  Hippuric  acid,  however,  shows  no 
"activating"  effect  on  the  digestion  of  starch  by  saliva  and  purified  pan- 
creatic amylase. 

It  was  thought  best  to  determine  whether  this  failure  to  "activate"  the 
enzyme  might  be  due  to  the  hydrolysis  of  the  hippuric  acid  during  digestion, 
forming  glycine  and  benzoic  acid.  By  a  colorimetric  test,  using  stand- 
ardized buffer  solutions,13  and  bromothymol  blue  as  indicator,  it  was 
found  that  the  hydrogen-ion  concentration  was  the  same  before  and  after 
"  Ref.  11,  p.  76. 


—  •'  12 

digestion  and,  therefore,  that  hydrolysis  of  hippuric  acid  did  not  occur  to 
any  appreciable  extent.  Table  IV  shows  a  typical  experiment  in  which 
the  influence  of  glycine  alone,  of  benzoic  acid  alone,  of  a  mixture  of  equal 
weights  of  glycine  and  benzoic  acid,  and  of  equimolar  amounts  of  each, 
is  tested. 

TABLE  IV 

COMPARISON  OF  THE  INFLUENCE  OF  BENZOIC  ACID  PLUS  GLYCINE  ON  THE  HYDROLYSIS 
OF  STARCH  BY  PURIFIED  PANCREATIC  AMYLASE 

Material  added  Cuprous  oxide 

Mg.          '  Mg. 

None 288 

Benzoic  acid  50 280 

Glycine  50 331 

Benzoic  acid  50  +  glycine  50 335 

Benzoic  acid  0.0066  M,  +  glycine  0.0066  M 326 

This  experiment  shows  the  usual  "activation"  with  glycine,  independent 
of  the  presence  of  the  benzoic  acid. 

The  effect  of  indole  and  guanidine  on  the  hydrolysis  of  starch  by  purified 
pancreatic  amylase,  and  the  influence  of  these  groups  in  the  amino  acids, 
were  next  tested.  It  was  found  that  guanidine,  like  arginine,  reacts  with 
Fehling  solution  so  that  a  test  upon  saccharogenic  activity  could  not 
be  made.  The  influence  of  these  substances  on  the  amyloclastic  activity 
of  the  enzyme  was  studied,  instead,  by  a  method  based  on  that  of  Wohl- 
gemuth,14  and  used  in  previous  work  in  this  Laboratory.15  The  results 
indicate  that  guanidine  has  no  effect  on  the  amyloclastic  activity  of  the 
enzyme,  while  indole  shows  an  inhibitory  effect.  This  accords  with  the 
observation  that  arginine  has  a  favorable  effect  upon  the  amyloclastic 
action  of  the  enzyme  while  tryptophane  has  not.8  In  view  of  these  ex- 
periments, one  might  reason  that  the  indifferent  behavior  of  tryptophane, 
compared  with  the  activating  influence  of  most  of  the  other  amino  acids, 
is  explainable  on  the  hypothesis  that  the  favorable  effect  of  its  alanine 
group  is  offset  by  the  inhibitory  influence  of  the  indole  radical.  However, 
the  explanation  that  tryptophane  may  be  so  bound  in  the  enzyme  mole- 
cule that  it  is  not  liberated  by  hydrolysis  until  after  the  amyloclastic 
activity  of  the  enzyme  has  been  injured,  seems  more  consistent,  when 
studied  in  the  light  of  further  investigation.  When  a  comparison  was 
made  of  the  influence  of  alanine,  phenylalanine  and  tryptophane  on  the 
saccharogenic  activity  of  the  amylase,  the  result  showed  that  tryptophane 
acts  like  most  of  the  other  amino  acids  in  increasing  the  saccharogenic 
activity  of  the  enzyme.  The  results  of  a  typical  comparison  are  given  in 
Table  V. 

14  Wohlgemuth,  Biochem.  Z.,  9,  1  (1908). 

16  Sherman  and  Thomas,  /.  Am.  Chem.  Soc.,  37,  634  (1915).     Ref.  8. 


13 

TABUJ  V 

INFLUENCE  OP  AI,ANINE,  PHENYLALANINE,  TRYPTOPHANE  AND  INDOLE  ON  THE 

HYDROLYSIS  OP  STARCH  BY  PURIFIED  PANCREATIC  AMYLASE 
Material  added        Cuprous  oxide  Material  added  Cuprous  oxide 

0.005  M  Mg.  0.005  M  Mg. 

None 300 

Alanine 339  Tryptophane 338 

Phenylalanine 341  Indole 275 

Since  consistent  "activation"  is  obtained  with  tryptophane  as  with  other 
amino  acids  tested,  upon  the  saccharogenic  property  of  the  amylase,  it 
is  evident  that  in  this  case  the  added  substance  affects  the  amyloclastic 
and  saccharogenic  activities  differently.  With  the  other  amino  acids 
here  tested,  the  influence  has  been  the  same  towards  the  two  properties 
of  the  enzyme;  but  tryptophane  is  not  unique  in  augmenting  the  sacchar- 
ogenic but  not  the  amyloclastic  activity,  for  in  other  experiments  in  this 
Laboratory  the  same  has  been  found  with  respect  to  lysine.16 

This  effect  is  not  due  to  hydrogen-ion  concentration,  since  this  was 
constant  in  the  digestion  mixtures  used  in  all  experiments,  as  has  been 
stated  before,  and  since  previous  work17  has  shown  that,  for  optimum  ac- 
tivity of  the  amyloclastic  and  saccharogenic  properties  of  pancreatic  amy- 
lase, the  range  of  hydrogen-ion  concentration  is  the  same.  However, 
since  the  enzyme  molecule  is  in  all  probability  of  a  protein  nature,  the 
tryptophane  may  be  so  bound  in  the  molecule  that  it  would  not  be  lib- 
erated until  after  the  amyloclastic  activity  of  the  enzyme  was  lost  and, 
therefore,  any  added  tryptophane  would  not  affect  the  amyloclastic  prop- 
erty, but  still  might  protect  the  enzyme  from  further  hydrolytic  changes 
whereby  its  saccharogenic  activity  would  be  affected. 

Summary 

The  favorable  effect  reported  by  Rockwood  to  be  exerted  by  several 
types  of  organic  compounds  upon  the  activity  of  amylolytic  enzymes,  in 
consequence  of  which  he  applied  the  term  auxo-amylases  to  these  com- 
pounds, appears  to  have  been  due  in  most  if  not  all  cases,  other  than  those 
of  natural  amino  acids,  to  hydrogen-ion  or  salt  effects,  rather  than  to  the 
organic  structure  of  the  compounds. 

Tested  upon  salivary  or  pancreatic  amylase  in  the  presence  of  favorable 
concentrations  of  chloride,  phosphate  and  hydrogen  ions,  methyl  and 
ethyl  amine  hydrochlorides,  aniline  sulfate,  benzoic  acid,  benzamide, 
anthranilic  acid  and  hippuric  acid  failed  to  show  any  favorable  effect 
upon  the  activity  of  the  enzyme.  Hence,  it  appears  probable  that  none 
of  the  types  of  compounds  illustrated  by  these  substances  has  any  ac- 
tivating influence  upon  salivary  or  pancreatic  amylase  which  can  properly 
be  attributed  to  their  organic  structure. 

16  Sherman  and  Caldwell,  J.  Am.  Chem.  Soc.,  44,  2926  (1922). 

17  Sherman  and  Schlesinger,  ibid.,  35,  1784  (1913). 


14 

Previous  findings  regarding  the  favorable  influence  of  several  amino 
acids  resulting  from  protein  hydrolysis  have  been  confirmed  and  extended. 
This  influence  may  be  attributed  either  to  a  direct  "activating"  effect  de- 
pendent upon  the  structural  nature  of  these  substances  as  a-amino 
acids,  or  to  conservation  of  the  enzyme  by  retarding  its  hydrolysis.  While 
the  hypothesis  of  direct  "activation"  exerted  by  a-amino  compounds  as 
such  is  not  disproved,  the  results  of  tests  with  hippuric  acid  fail  to  give  it 
any  support.  The  results  obtained  in  this  investigation  can  all  be  explained 
on  the  basis  of  the  conservation  hypothesis  alone. 


VITA 

Nellie  M.  Naylor  was  born  at  Clear  Lake,  Iowa,  March  20,  1885.  She 
prepared  for  college  at  the  Clear  Lake  High  School,  and  entered  Iowa 
State  College  in  1902,  taking  two  years  of  collegiate  work  there  and  later, 
two  years  at  the  State  University.  She  received  the  degeree  of  Bachelor  of 
Arts  from  Iowa  State  University  in  1908.  She  was  an  Assistant  in  Chem- 
istry at  Iowa  State  College  from  1909  to  1911,  and  an  Instructor  in  Chem- 
istry from  1911  to  1920.  She  was  a  graduate  student  at  the  University  of 
Chicago  during  the  summers  of  1910,  1911  and  1919,  and  received  the 
degree  of  Master  of  Science  at  Iowa  State  College  in  June,  1918.  She  has 
been  a  graduate  student  in  the  School  of  Pure  Science  Columbia  University 
during  the  academic  years  1920-1921  and  1921-1922. 

She  was  co-author  with  Dr.  R.  R.  Renshaw  of  a  paper  entitled  "Dyes 
containing  the  Furane  Cycle,"  published  in  the  Journal  of  the  American 
Chemical  Society  44,  862  (1922). 


14  DAY  USE 

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